Method for expression of small RNA molecules within a cell

ABSTRACT

The invention provides methods and compositions for the expression of small RNA molecules within a cell using a lentiviral vector. The methods can be used to express doubles stranded RNA complexes. Small interfering RNA (siRNA) can be expressed using the methods of the invention within a cell, which is capable of down regulating the expression of a target gene through RNA interference. A variety of cells can be treated according to the methods of the invention including embryos, embryogenic stem cells, allowing for the generation of transgenic animals or animals constituted partly by the transduced cells that have a specific gene or a group of genes down regulated.

REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 15/391,189, filed Dec. 27, 2016, which is a continuation ofU.S. patent application Ser. No. 14/622,064, filed Feb. 13, 2015, nowU.S. Pat. No. 9,551,011, which is a continuation of U.S. patentapplication Ser. No. 13/750,903, filed Jan. 25, 2013, now U.S. Pat. No.8,969,076, which is a continuation of U.S. patent application Ser. No.12/795,581, now U.S. Pat. No. 8,361,787, filed Jun. 7, 2010, which is acontinuation of U.S. patent application Ser. No. 10/243,816, now U.S.Pat. No. 7,732,193, filed Sep. 13, 2002, which in turn claims priorityunder 35 U.S.C. § 119(e) to U.S. Provisional Application No. 60/322,031,filed Sep. 13, 2001, U.S. Provisional Application No. 60/347,782, filedJan. 9, 2002, U.S. Provisional Application No. 60/389,592, filed Jun.18, 2002, and U.S. Provisional Application No. 60/406,436, filed Aug.27, 2002. All of the aforementioned priority applications are herebyincorporated by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under Grant NumbersGM039458, AI039975, and AI055281 awarded by the National Institutes ofHealth. The United States Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to methods for altering geneexpression in a cell or an animal using viral constructs engineered todeliver an RNA molecule. In a more specific aspect, a viral construct isused to deliver double-stranded RNA molecules that can be used todown-regulate or modulate gene expression.

Description of the Related Art

RNA interference (RNAi) or silencing is a recently discovered phenomenon(A. Fire et al., Nature 391, 806 (1998); C. E. Rocheleau et al. Cell 90,707 (1997)). Small interfering RNAs (“siRNAs”) are double-stranded RNAmolecules that inhibit the expression of a gene with which they sharehomology. siRNAs have been used as a tool to down regulate theexpression of specific genes in a variety of cultured cells as well asin invertebrate animals. A number of such approaches have been reviewedrecently (P. D. Zamore Science 296, 1265 (2002)); however, suchapproaches have limitations. For example, no technique prior to theinvention described herein allows for the generation of transgenicmammal having a specific gene down regulated through RNA interference.Similarly, there is a need for more robust methods for the introductionof small RNA molecules with regulatory function. The invention providedherein addresses these and other limitations in the field of RNAmediated gene regulation.

SUMMARY OF THE INVENTION

The invention relates generally to methods to express within a cell anRNA molecule or molecules. These methods can be used with a wide varietyof cell types. RNA molecules can be expressed within a cell for avariety of purposes. For example, and without limitation, RNA moleculescan serve as markers within a cell, can act as antisenseoligonucleotides or ribozymes for regulating gene expression, and canserve to down regulate genes through RNA interference.

In one aspect, the invention provides retroviral constructs for theexpression of an RNA molecule or molecules within a cell. The constructspreferably comprise a nucleic acid having the R and U5 sequences from a5′ lentiviral long terminal repeat (LTR), a self-inactivating lentiviral3′ LTR, and a RNA Polymerase III (pol III) promoter. The retroviralconstructs preferably comprise an RNA coding region operably linked tothe RNA Polymerase III promoter. The RNA coding region preferablycomprises a DNA sequence that can serve as a template for the expressionof a desired RNA molecule.

The RNA coding region can be immediately followed by a pol IIIterminator sequence which directs the accurate and efficient terminationof RNA synthesis by pol III. The pol III terminator sequences generallycomprise 4 or more consecutive T residues. In a preferred embodiment, acluster of 5 consecutive Ts is used as the terminator by which pol IIItranscription is stopped at second or third T of the DNA template. As aresult, only 2 to 3 U residues are added to the 3′ end of the RNA thatis synthesized from the RNA coding region.

A variety of pol III promoters can be used with the invention, includingfor example, the promoter fragments derived from H1 RNA genes or U6 snRNA genes of human or mouse origin or from any other species. Inaddition, pol III promoters can be modified/engineered to incorporateother desirable properties such as to be inducible by small chemicalmolecules either ubiquitously or in a tissue-specific manner, forexample, one activated with tetracycline or IPTG (lacI system).

The pol III promoter, RNA template region and pol III terminatortogether may comprise an “RNA cassette” or “RNA expression cassette.” Ifthe RNA is a small inhibitory RNA (siRNA), the expression cassette maybe termed an “siRNA expression cassette.”

In one embodiment, the RNA coding region encodes a self-complementaryRNA molecule having a sense region, an antisense region and a loopregion. Such an RNA molecule when expressed desirably forms a “hairpin”structure. The loop region is generally between about 2 and about 10nucleotides in length. In a preferred embodiment, the loop region isfrom about 6 and about 9 nucleotides in length. In one such embodimentof the invention, the sense region and the antisense region are betweenabout 15 and about 30 nucleotides in length.

In one embodiment, the RNA coding region is operably linked downstreamto an RNA Polymerase III promoter such that the RNA coding sequence canbe precisely expressed without any extra non-coding nucleotides presentat 5′ end. In this way an RNA sequence can be expressed that isidentical to a target sequence at the 5′ end. The synthesis of the RNAcoding region is ended at the terminator site. In one preferredembodiment the terminator consists of five consecutive T residues.

In another aspect of the invention, the retroviral vector can comprisemultiple RNA coding regions. In one embodiment, the retroviral constructcomprises a first RNA pol III promoter, a first coding region encoding afirst RNA molecule operably linked to the first RNA pol III promoter, asecond RNA pol III promoter and a second RNA coding region operablylinked to the second RNA pol III promoter. Preferably, the second RNAcoding region encodes an RNA molecule that is substantiallycomplementary to the RNA molecule encoded by the first RNA codingregion, such that the two RNA molecules can form a double-strandedstructure when expressed. The methods of invention also include multipleRNA coding regions that encode hairpin-like self-complementary RNAmolecules or other non-hairpin molecules.

In yet another embodiment of the invention, the retroviral constructcomprises a first RNA pol III promoter operably linked to a first RNAcoding region, and a second RNA pol III promoter operably linked to thesame first RNA coding region in the opposite direction, such thatexpression of the RNA coding region from the first RNA pol III promoterresults in a synthesis of a first RNA molecule as the sense strand andexpression of the RNA coding region from the second RNA pol III promoterresults in synthesis of a second RNA molecule as an antisense strandthat is substantially complementary to the first RNA molecule. In onesuch embodiment, both RNA Polymerase III promoters are separated fromthe RNA coding region by termination sequences, preferably terminationsequences having five consecutive T residues.

According to one embodiment of the invention, the 5′ LTR sequences inthe retroviral construct are derived from HIV. The retroviral constructmay also comprise a woodchuck hepatitis virus enhancer element sequenceand/or a tRNA amber suppressor sequence.

In another embodiment of the invention, the self-inactivating 3′ LTR isa U3 element with a deletion of its enhancer sequence. In yet anotherembodiment, the self-inactivating 3′ LTR is a modified HIV 3′ LTR.

The recombinant retroviral construct can be pseudotyped, for examplewith the vesicular stomatitits virus envelope glycoprotein.

In another aspect of the invention, expression of the RNA coding regionresults in the down regulation of a target gene. Preferably the targetgene comprises a sequence that is at least about 90% identical with theRNA coding region, more preferably at least about 95% identical, andeven more preferably at least about 99% identical.

According to a further aspect of the invention, the viral construct alsocomprises a nucleotide sequence encoding a gene of interest. The gene ofinterest is preferably operably linked to a Polymerase II promoter. Sucha construct also can contain, for example, an enhancer sequence operablylinked with the Polymerase II promoter.

A variety of Polymerase II promoters can be used with the invention,including for example, the CMV promoter. The RNA Polymerase II promoterthat is chosen can be a ubiquitous promoter, capable of drivingexpression in most tissues, for example, the human Ubiquitin-C promoter,CMV β-actin promoter or PGK promoter. In other embodiments the RNAPolymerase II promoter is a tissue-specific promoter.

In one embodiment, the gene of interest is a marker or reporter gene,that can be used to verify that the vector was successfully transfectedor transduced and its sequences expressed. In one such embodiment, thegene of interest is a fluorescent reporter gene, for example, the GreenFluorescent Protein. In yet another embodiment, the gene of interest isa drug resistant gene which can be used to select the cells that aresuccessfully transduced. For example, the drug resistant gene can be thezeocin resistant gene (zeo). The gene of interest also can be a hybridof a drug resistant gene and a fluorescent reporter gene, such as azeo/gfp fusion. In another embodiment, the gene of interest encodes aprotein factor that can regulate the transcription activity of induciblepol III promoters. In one of such embodiment, the gene of interest istetR (repressor for tet operon) which regulates tetracycline responsivepol Ill promoters.

It is another aspect of the invention to provide methods for expressingan RNA molecule or molecules within a cell. According to the invention,a packaging cell line is transfected with a retroviral construct of theinvention, recombinant retroviral particles are recovered from thepackaging cell line; and a target cell is infected with the recombinantretrovirus particles.

In one embodiment of the invention, the target cell is an embryoniccell. An embryonic cell may be, for example, a single cell embryo orembryonic cells from within an early-stage embryo. In another embodimentof the invention, the target cell is an embryogenic stem cell. When thetarget cell is an embryonic cell, in one embodiment the embryonic cellis infected by injecting the recombinant retrovirus between the zonapellucida and the cell membrane of the embryonic cell. In anotherembodiment, the embryonic cell is infected by removing the zonapellucida and incubating the cell in solution containing the recombinantretrovirus. In such an embodiment, the zona pellucida can be removed,for example, by enzymatic digestion.

When the target cell is an embryonic cell or an embryogenic stem cell,the cell may be transplanted in a pseudopregnant female to generate atransgenic animal.

The methods of the invention also can be used with a variety of primaryex vivo normal or diseased cells or cells adapted in various tissueculture conditions from human, mouse and other vertebrates, including,without limitation, stem or precursor cells for the hematopoieticsystem, central nerve system cells, cells with regenerative capacitiesfrom a variety of other tissues and organs, dendritic cells and otherdeveloping and mature myeloid and lymphoid cells, and cancer cellsderived from different cell lineages.

In a particular embodiment, the target cell is an embryonic cell of abird within an egg. The embryonic cell of a bird is preferably infectedby contacting the embryonic blastodisc of the bird egg with retroviralparticles.

In yet another embodiment, the target cell is a fish egg. The fish eggis preferably infected by delivering the retroviral particles to thespace between the chorion and the cell membrane of the fish egg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic diagram of a retroviral vector carrying anexpression cassette for RNA expression, termed “RNA cassette” and a“Marker Gene” or gene of interest. The RNA expression cassette can beembedded at any permissible sites of the retroviral construct either assingle copy or multiple tandem copies. In addition, although notindicated in the figure, more than one RNA expression cassette may bepresent in the retroviral construct. FIG. 1B shows a similar constructin which the RNA expression cassettes flank a marker gene.

FIG. 2 shows a schematic view of an RNA expression cassette comprisingan RNA Polymerase III promoter 100 linked to an RNA coding region110-130 and a terminator sequence 140. The RNA coding region comprises asense region 110, a loop region 120, and an antisense region 130,

FIG. 3 shows a schematic view of an RNA expression cassette having anRNA Polymerase III promoter 100 linked to a first RNA coding region 110and a first terminator sequence 140 and a second RNA polymerase IIIpromoter 105 linked to a second RNA coding region 115 and a secondterminator 145.

FIG. 4 shows a schematic view of an RNA expression cassette having afirst RNA Polymerase III promoter 100 linked to an RNA coding region 110and a first terminator sequence 145. The expression cassette has asecond RNA polymerase III promoter 105 linked to the RNA coding region115, the same sequence as 110 in reverse, and a second terminator 140.

FIG. 5. Schematic illustration of a lacZ siRNA encoding lentiviralvector. 5′LTR: an HIV based lentiviral vector 5′ LTR; F: an HIV Flapelement; pol III: a human H1-RNA pol III promoter (−240 to −8); siRNA: alacZ specific small hairpin RNA coding region and its structure anddetailed sequence are illustrated below. UbiC: an internal humanubiquitinC promoter; GFP: a GFP marker gene driven by UbiC promoter. W:a woodchuck RNA regulatory element. 3′LTR: an HIV based selfinactivating lentiviral 3′ LTR.

FIG. 6. A lacZ specific siRNA encoded by a lentiviral vector canefficiently inhibit the expression of lacZ reporter gene in virustransduced mammalian cells. MEF: mouse embryonic fibroblasts; HEK293:human embryonic kidney cells. Both of the test cell lines harbor lacZand firefly luciferase reporter genes, and the expression levels of thereporter genes can be measured by chemiluminescent assays. Ctrl: theratio of lacZ activity versus Luc activity of the uninfected parentalcells, which was arbitrarily set to 1. Transduced: the specificinhibition of lacZ expression calculated as the reduction of lacZ to Lucratio.

FIG. 7. Transgenic animals that express a lacZ specific siRNA moleculeencoded by a lentiviral vector can successfully suppress the expressionof the ubiquitous lacZ reporter gene in a ROSA26+/− background. ROSA1-6:the lacZ activities in the limb tissues of six E17.5 ROSA26+/− embryoswhich served as positive controls. The difference in lacZ activitybetween individual ROSA26+/− embryos may result from variable proteinextraction efficiency. TG1-4: the lacZ activities in the limb tissues offour E17.5 transgenic embryos expressing a lentiviral vector-encodedlacZ siRNA molecule in ROSA+/− background. WT1-6: lacZ activity in thelimb tissues of six E17.5 C57Bl/6 wildtype embryos, included as thenegative control. The background levels of endogenous beta-galactosidaseactivity are general below 1,000 LU/ug, thus the columns are notvisible.

FIG. 8 shows a schematic illustration of a Tet-inducible lacZ siRNAlentiviral vector. A Tet repressor gene (TetR; SEQ ID NO: 5) is theunder the control human UbiquitinC promoter and its expression can bemonitored by the downstream GFP marker coupled by IRES element (internalribosomal entry site). The anti-lacZ siRNA cassette is driven by aTet-inducible pol III promoter derived from human U6-promoter (−328 to+1) containing a single TetR binding site (TetO1) between the PSE andTATA box (SEQ ID NO: 4). In the absence of tetracycline, TetR binds tothe promoter and its expression is repressed. Upon the addition oftetracycline, TetR is moved from the promoter and transcription starts.

FIG. 9 shows the results of an experiment that demonstrated that aTet-inducible siRNA expression cassette can regulate gene expression inresponse to Doxycycline treatment. lacZ and luciferase double expressingHEK293 cells (293Z+Luc) were transduced with a lentiviral vectorcarrying a Tet-inducible lacZ-siRNA cassette and a Tet repressor underthe control of a UbiquitinC promoter (FIG. 8). The transduced cells weretreated with 10 ug/ml Doxycycline (Plus Dox) for 48 hr or without theDoxycycline treatment as a control (No Dox). LacZ and luciferaseactivities were measured as described in the previous figures. Therelative suppression activity is calculated as the ratio of lacZ versusluciferase and No Dox control was arbitrarily set to 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The inventors have previously identified a method for introducing atransgene of interest into a cell or animal. This technique is describedin U.S. provisional patent application No. 60/322,031 filed on Sep. 13,2001 and U.S. provisional patent application No. 60/347,782 filed onJan. 9, 2002, the entire contents of which are incorporated herein byreference.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Any methods, devices andmaterials similar or equivalent to those described herein can be used inthe practice of this invention.

By “transgene” is meant any nucleotide sequence, particularly a DNAsequence, that is integrated into one or more chromosomes of a host cellby human intervention, such as by the methods of the present invention.In one embodiment, a transgene is an “RNA coding region.” In anotherembodiment the transgene comprises a “gene of interest.” In otherembodiments the transgene can be a nucleotide sequence, preferably a DNAsequence, that is used to mark the chromosome where it has integrated.In this situation, the transgene does not have to comprise a gene thatencodes a protein that can be expressed.

A “gene of interest” is a nucleic acid sequence that encodes a proteinor other molecule that is desirable for integration in a host cell. Inone embodiment, the gene of interest encodes a protein or other moleculethe expression of which is desired in the host cell. In this embodiment,the gene of interest is generally operatively linked to other sequencesthat are useful for obtaining the desired expression of the gene ofinterest, such as transcriptional regulatory sequences.

A “functional relationship” and “operably linked” mean, withoutlimitation, that the gene is in the correct location and orientationwith respect to the promoter and/or enhancer that expression of the genewill be affected when the promoter and/or enhancer is contacted with theappropriate molecules.

An “RNA coding region” is a nucleic acid that can serve as a templatefor the synthesis of an RNA molecule, such as an siRNA. Preferably, theRNA coding region is a DNA sequence.

A “small interfering RNA” or “siRNA” is a double-stranded RNA moleculethat is capable of inhibiting the expression of a gene with which itshares homology. In one embodiment the siRNA may be a “hairpin” orstem-loop RNA molecule, comprising a sense region, a loop region and anantisense region complementary to the sense region. In other embodimentsthe siRNA comprises two distinct RNA molecules that are non-covalentlyassociated to form a duplex.

The term “transgenic” is used herein to describe the property ofharboring a transgene. For instance, a “transgenic organism” is anyanimal, including mammals, fish, birds and amphibians, in which one ormore of the cells of the animal contain nucleic acid introduced by wayof human intervention, such as by the methods described herein. In atransgenic animal that comprises a transgene that encodes a gene ofinterest, the transgene typically causes the cell to express oroverexpress a recombinant protein. However, according to the methods ofthe invention, expression of an RNA coding region can be used to downregulate the expression of a particular gene through antisense or RNAinterference mechanisms.

The terms “founder,” “founder animal” and “founder line” refer to thoseanimals that are mature products of the embryos or oocytes to which thetransgene was added, i.e. those animals that grew from the embryos oroocytes into which DNA was inserted.

The terms “progeny” and “progeny of the transgenic animal” refer to anyand all offspring of every generation subsequent to the originallytransformed animal.

The term “animal” is used in its broadest sense and refers to allanimals including mammals, birds, fish, reptiles and amphibians.

The term “mammal” refers to all members of the class Mammalia andincludes any animal classified as a mammal, including humans, domesticand farm animals, and zoo, sports or pet animals, such as mouse, rabbit,pig, sheep, goat, cattle and higher primates.

The term “oocyte” refers to a female gamete cell and includes primaryoocytes, secondary oocytes and mature, unfertilized ovum. As usedherein, the term “egg” when used in reference to a mammalian egg, meansan oocyte surrounded by a zona pellucida. The term “zygote” refers to afertilized ovum. The term “embryo” broadly refers to an animal in theearly stages of development.

“Perivitelline space” refers to the space located between the zonapellucida and the cell membrane of a mammalian egg or embryonic cell.

“Target cell” or “host cell” means a cell that is to be transformedusing the methods and compositions of the invention.

“Lentivirus” refers to a genus of retroviruses that are capable ofinfecting dividing and non-dividing cells. Several examples oflentiviruses include HIV (human immunodeficiency virus; including HIVtype 1, and HIV type 2), the etiologic agent of the human acquiredimmunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis(visna) or pneumonia (maedi) in sheep, the caprinearthritis-encephalitis virus, which causes immune deficiency, arthritis,and encephalopathy in goats; equine infectious anemia virus, whichcauses autoimmune hemolytic anemia, and encephalopathy in horses; felineimmunodeficiency virus (FIV), which causes immune deficiency in cats;bovine immune deficiency virus (BIV), which causes lymphadenopathy,lymphocytosis, and possibly central nervous system infection in cattle;and simian immunodeficiency virus (SIV), which cause immune deficiencyand encephalopathy in sub-human primates.

A lentiviral genome is generally organized into a 5′ long terminalrepeat (LTR), the gag gene, the pol gene, the env gene, the accessorygenes (nef, vif, vpr, vpu) and a 3′ LTR. The viral LTR is divided intothree regions called U3, R and U5. The U3 region contains the enhancerand promoter elements. The U5 region contains the polyadenylationsignals. The R (repeat) region separates the U3 and U5 regions andtranscribed sequences of the R region appear at both the 5′ and 3′ endsof the viral RNA. See, for example, “RNA Viruses: A Practical Approach”(Alan J. Cann, Ed., Oxford University Press, (2000)), O Narayan andClements J. Gen. Virology 70:1617-1639 (1989), Fields et al. FundamentalVirology Raven Press. (1990), Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma I M. J Virol. 72(10):8150-7 (1998), and U.S. Pat. No.6,013,516.

Lentiviral vectors are known in the art, including several that havebeen used to transfect hematopoietic stem cells. Such vectors can befound, for example, in the following publications, which areincorporated herein by reference: Evans J T et al. Hum Gene Ther 1999;10:1479-1489; Case S S, Price M A, Jordan C T et al. Proc Natl Acad SciUSA 1999; 96:2988-2993; Uchida N, Sutton R E, Friera A M et al. ProcNatl Acad Sci USA 1998; 95:11939-11944; Miyoshi H, Smith K A, Mosier D Eet al. Science 1999; 283:682-686; Sutton R E, Wu H T, Rigg R et al.Human immunodeficiency virus type 1 vectors efficiently transduce humanhematopoietic stem cells. J Virol 1998; 72:5781-5788.

“Virion,” “viral particle” and “retroviral particle” are used herein torefer to a single virus comprising an RNA genome, pol gene derivedproteins, gag gene derived proteins and a lipid bilayer displaying anenvelope (glyco)protein. The RNA genome is usually a recombinant RNAgenome and thus may contain an RNA sequence that is exogenous to thenative viral genome. The RNA genome may also comprise a defectiveendogenous viral sequence.

A “pseudotyped” retrovirus is a retroviral particle having an envelopeprotein that is from a virus other than the virus from which the RNAgenome is derived. The envelope protein may be from a differentretrovirus or from a non-retroviral virus. A preferred envelope proteinis the vesicular stomatitius virus G (VSV G) protein. However, toeliminate the possibility of human infection, viruses can alternativelybe pseudotyped with ecotropic envelope protein that limit infection to aspecific species, such as mice or birds. For example, in one embodiment,a mutant ecotropic envelope protein is used, such as the ecotropicenvelope protein 4.17 (Powell et al. Nature Biotechnology 18(12):1279-1282 (2000)).

The term “provirus” is used to refer to a duplex DNA sequence present ina eukaryotic chromosome that corresponds to the genome of an RNAretrovirus. The provirus may be transmitted from one cell generation tothe next without causing lysis or destruction of the host cell.

A “self-inactivating 3′ LTR” is a 3′ long terminal repeat (LTR) thatcontains a mutation, substitution or deletion that prevents the LTRsequences from driving expression of a downstream gene. A copy of the U3region from the 3′ LTR acts as a template for the generation of bothLTR's in the integrated provirus. Thus, when the 3′ LTR with aninactivating deletion or mutation integrates as the 5′ LTR of theprovirus, no transcription from the 5′ LTR is possible. This eliminatescompetition between the viral enhancer/promoter and any internalenhancer/promoter. Self-inactivating 3′ LTRs are described, for example,in Zufferey et al. J. Virol. 72:9873-9880 (1998), Miyoshi et al. J.Virol. 72:8150-8157 and Iwakuma et al. Virology 261:120-132 (1999).

The term “RNA interference or silencing” is broadly defined and includesall posttranscriptional and transcriptional mechanisms of RNA mediatedinhibition of gene expression, such as those described in (P. D. ZamoreScience 296, 1265 (2002)).

In one aspect of the invention, a recombinant retrovirus is used todeliver a transgene comprising an RNA coding region of interest to atarget cell. Preferably the target cell is a mammalian cell. The cellmay be a primary cell, or may be a cultured cell, for example an withoutlimitation an HEK, CHO, COS, MEF, 293 cell. In one embodiment the targetcell is an oocyte or an embryonic cell, more preferably a one-cellembryo. The RNA coding region and any associated genetic elements arethus integrated into the genome of the target cell as a provirus. Whenthe target cell is an embryo, the cell may then be allowed to developinto a transgenic animal by methods well known in the art.

The recombinant retrovirus used to deliver the RNA coding region ispreferably a modified lentivirus, and thus is able to infect bothdividing and non-dividing cells. The recombinant retrovirus preferablycomprises a modified lentiviral genome that includes the transgene.Further, the modified lentiviral genome preferably lacks endogenousgenes for proteins required for viral replication, thus preventingundesired replication, such as replication in a resulting transgenicanimal. The required proteins are preferably provided in trans in thepackaging cell line during production of the recombinant retrovirus, asdescribed below.

In another embodiment, the recombinant retrovirus used to deliver theRNA coding region is a modified Moloney virus, for example a MoloneyMurine Leukemia Virus. In a further embodiment, the virus is a MurineStem Cell Virus (Hawley, R. G., et al. (1996) Proc. Natl. Acad. Sci. USA93:10297-10302; Keller, G., et al. (1998) Blood 92:877-887; Hawley, R.G., et al. (1994) Gene Ther. 1:136-138). The recombinant retrovirus alsocan be a hybrid virus such as that described in Choi, J K; Hoanga, N;Vilardi, A M; Conrad, P; Emerson, S G; Gewirtz, A M. (2001) HybridHIV/MSCV LTR Enhances Transgene Expression of Lentiviral Vectors inHuman CD34+ Hematopoietic Cells. Stem Cells 19, No. 3, 236-246.

In the preferred embodiment the transgene is incorporated into a viralconstruct that comprises an intact retroviral 5′ LTR and aself-inactivating 3′ LTR. The viral construct is preferably introducedinto a packaging cell line that packages viral genomic RNA based on theviral construct into viral particles with the desired host specificity.Viral particles are collected and used to infect the host cell. Each ofthese aspects is described in detail below.

The Viral Construct

The viral construct is a nucleotide sequence that comprises sequencesnecessary for the production of recombinant retrovirus in a packagingcell. In one embodiment the viral construct additionally comprisesgenetic elements that allow for the desired expression of an RNAmolecule or gene of interest in the host.

Generation of the viral construct can be accomplished using any suitablegenetic engineering techniques well known in the art, including, withoutlimitation, the standard techniques of PCR, oligonucleotide synthesis,restriction endonuclease digestion, ligation, transformation, plasmidpurification, and DNA sequencing, for example as described in Sambrooket al. (Molecular Cloning: A Laboratory Manual. Cold Spring HarborLaboratory Press, N.Y. (1989)), Coffin et al. (Retroviruses. Cold SpringHarbor Laboratory Press, N.Y. (1997)) and “RNA Viruses: A PracticalApproach” (Alan J. Cann, Ed., Oxford University Press, (2000)).

The viral construct may incorporate sequences from the genome of anyknown organism. The sequences may be incorporated in their native formor may be modified in any way. For example, the sequences may compriseinsertions, deletions or substitutions. In the preferred embodiment theviral construct comprises sequences from a lentivirus genome, such asthe HIV genome or the SIV genome.

The viral construct preferably comprises sequences from the 5′ and 3′LTRs of a lentivirus. More preferably the viral construct comprises theR and U5 sequences from the 5′ LTR of a lentivirus and an inactivated orself-inactivating 3′ LTR from a lentivirus. The LTR sequences may be LTRsequences from any lentivirus from any species. For example, they may beLTR sequences from HIV, SIV, FIV or BIV. Preferably the LTR sequencesare HIV LTR sequences. The virus also can incorporate sequences from MMVor MSCV.

The viral construct preferably comprises an inactivated orself-inactivating 3′ LTR. The 3′ LTR may be made self-inactivating byany method known in the art. In the preferred embodiment the U3 elementof the 3′ LTR contains a deletion of its enhancer sequence, preferablythe TATA box, Sp1 and NF-kappa B sites. As a result of theself-inactivating 3′ LTR, the provirus that is integrated into the hostcell genome will comprise an inactivated 5′ LTR.

Optionally, the U3 sequence from the lentiviral 5′ LTR may be replacedwith a promoter sequence in the viral construct. This may increase thetiter of virus recovered from the packaging cell line. An enhancersequence may also be included. Any enhancer/promoter combination thatincreases expression of the viral RNA genome in the packaging cell linemay be used. In the preferred embodiment the CMV enhancer/promotersequence is used (U.S. Pat. No. 5,168,062; Karasuyama et al J. Exp. Med.169:13 (1989).

The viral construct also comprises a transgene. The transgene, may beany nucleotide sequence, including sequences that serve as markers forthe provirus. Preferably the transgene comprises one or more RNA codingregions and/or one or more genes of interest. Schematic diagrams ofexemplary retroviral constructs are shown in FIGS. 1A and 1B.

In the preferred embodiment the transgene comprises at least one RNAcoding region. Preferably the RNA coding region is a DNA sequence thatcan serve as a template for the expression of a desired RNA molecule inthe host cell. In one embodiment, the viral construct comprises two ormore RNA coding regions.

The viral construct also preferably comprises at least one RNAPolymerase III promoter. The RNA Polymerase III promoter is operablylinked to the RNA coding region and can also be linked to a terminationsequence. In addition, more than one RNA Polymerase III promoter may beincorporated.

RNA Polymerase III promoters are well known to one of skill in the art.A suitable range of RNA Polymerase III promoters can be found, forexample, in Paule and White. Nucleic Acids Research., Vol 28, pp1283-1298 (2000), which is hereby incorporated by reference in itsentirety. The definition of RNA Polymerase III promoters also includeany synthetic or engineered DNA fragment that can direct RNA PolymeraseIII to transcribe a downstream RNA coding sequence. Further, the RNAPolymerase III (Pol III) promoter or promoters used as part of the viralvector can be inducible. Any suitable inducible Pol III promoter can beused with the methods of the invention. Particularly suited Pol IIIpromoters include the tetracycline responsive promoters provided inOhkawa and Taira Human Gene Therapy, Vol. 11, pp 577-585 (2000) and inMeissner et al. Nucleic Acids Research, Vol. 29, pp 1672-1682 (2001),which are incorporated herein by reference.

In one embodiment the transgene comprises a gene of interest thatencodes a protein that is desirably expressed in one or more cells of atransgenic animal, for example, a reporter or marker protein. Preferablythe gene of interest is located between the 5′ LTR and 3′ LTR sequences.Further, the gene of interest is preferably in a functional relationshipwith other genetic elements, for example transcription regulatorysequences such as promoters and/or enhancers, to regulate expression ofthe gene of interest in a particular manner once the transgene isincorporated into the host genome. In certain embodiments, the usefultranscriptional regulatory sequences are those that are highly regulatedwith respect to activity, both temporally and spatially.

Preferably the gene of interest is in a functional relationship withinternal Polymerase II promoter/enhancer regulatory sequences. An“internal” promoter/enhancer is one that is located between the 5′ LTRand the 3′ LTR sequences in the viral construct and is operably linkedto the gene that is desirably expressed.

The Polymerase II promoter/enhancer may be any promoter, enhancer orpromoter/enhancer combination known to increase expression of a genewith which it is in a functional relationship.

The internal promoter/enhancer is preferably selected based on thedesired expression pattern of the gene of interest and the specificproperties of known promoters/enhancers. Thus, the internal promoter maybe a constitutive promoter. Non-limiting examples of constitutivepromoters that may be used include the promoter for ubiquitin, CMV (U.S.Pat. No. 5,168,062; Karasuyama et al J. Exp. Med. 169:13 (1989), β-actin(Gunning et al. Proc. Natl. Acad. Sci. USA 84:4831-4835 (1987) and pgk(see, for example, U.S. Pat. Nos. 4,615,974 and 5,104,795; Adra et al.Gene 60:65-74 (1987), Singer-Sam et al. Gene 32:409-417 (1984) andDobson et al. Nucleic Acids Res. 10:2635-2637 (1982)). Alternatively,the promoter may be a tissue specific promoter. Several non-limitingexamples of tissue specific promoters that may be used include 1ck (see,for example, Garvin et al. Mol. Cell Biol. 8:3058-3064 (1988) andTakadera et al. Mol. Cell Biol. 9:2173-2180 (1989)), myogenin (Yee etal. Genes and Development 7:1277-1289 (1993), and thy1 (Gundersen et al.Gene 113:207-214 (1992). In addition, promoters may be selected to allowfor inducible expression of the transgene. A number of systems forinducible expression using such a promoter are known in the art,including the tetracycline responsive system and the lacoperator-repressor system. It is also contemplated that a combination ofpromoters may be used to obtain the desired expression of the gene ofinterest. The skilled artisan will be able to select a promoter based onthe desired expression pattern of the gene in the resulting transgenicanimal.

An internal enhancer may also be present in the viral construct toincrease expression of the gene of interest. For example the CMVenhancer (Karasuyama et al J. Exp. Med. 169:13 (1989) may be used incombination with the chicken β-actin promoter (see, e.g., JP1990005890-A1). Again, one of skill in the art will be able to selectthe appropriate enhancer based on the desired expression pattern.

The gene of interest is not limited in any way and includes any genethat the skilled practitioner desires to have integrated and/orexpressed in a transgenic animal. For example, the gene of interest maybe one that encodes a protein that serves as a marker to identify cellscomprising the provirus. In other embodiments the gene of interestencodes a protein that modifies a physical characteristic of thetransgenic animal, such as a protein that modifies size, growth, ortissue composition. In another example the gene of interest may encode aprotein of commercial value that may be harvested from the transgenicanimal.

In addition, more than one gene of interest may be placed in functionalrelationship with the internal promoter. For example a gene encoding amarker protein may be placed after the primary gene of interest to allowfor identification of cells that are expressing the desired protein. Inone embodiment a fluorescent marker protein, preferably greenfluorescent protein (GFP), is incorporated into the construct along withthe gene of interest. If a second reporter gene is included, an internalribosomal entry site (IRES) sequence is also preferably included (U.S.Pat. No. 4,937,190). The IRES sequence may facilitate the expression ofthe reporter gene.

The viral construct may also contain additional genetic elements. Thetypes of elements that may be included in the construct are not limitedin any way and will be chosen by the skilled practitioner to achieve aparticular result. For example, a signal that facilitates nuclear entryof the viral genome in the target cell may be included. An example ofsuch a signal is the HIV-1 flap signal.

Further, elements may be included that facilitate the characterizationof the provirus integration site in the genome of the animal. Forexample, a tRNA amber suppressor sequence may be included in theconstruct.

In addition, the construct may contain one or more genetic elementsdesigned to enhance expression of the gene of interest. For example, awoodchuck hepatitis virus responsive element (WRE) may be placed intothe construct (Zufferey et al. J. Virol. 74:3668-3681 (1999); Deglon etal. Hum. Gene Ther. 11:179-190 (2000)).

A chicken β-globin insulator (Chung et al. Proc. Natl. Acad. Sci. USA94:575-580 (1997)) may also be included in the viral construct. Thiselement has been shown to reduce the chance of silencing the integratedprovirus in the transgenic animal due to methylation andheterochromatinization effects. In addition, the insulator may shieldthe internal enhancer, promoter and exogenous gene from positive ornegative positional effects from surrounding DNA at the integration siteon the chromosome.

Any additional genetic elements are preferably inserted 3′ of the geneof interest.

In a specific embodiment, the viral vector comprises: a cytomegalovirus(CMV) enhancer/promoter sequence; the R and U5 sequences from the HIV 5′LTR; the HIV-1 flap signal; an internal enhancer; an internal promoter;a gene of interest; the woodchuck hepatitis virus responsive element; atRNA amber suppressor sequence; a U3 element with a deletion of itsenhancer sequence; the chicken β-globin insulator; and the R and U5sequences of the 3′ HIV LTR.

The viral construct is preferably cloned into a plasmid that may betransfected into a packaging cell line. The preferred plasmid preferablycomprises sequences useful for replication of the plasmid in bacteria.

Production of Virus

Any method known in the art may be used to produce infectious retroviralparticles whose genome comprises an RNA copy of the viral constructdescribed above.

Preferably, the viral construct is introduced into a packaging cellline. The packaging cell line provides the viral proteins that arerequired in trans for the packaging of the viral genomic RNA into viralparticles. The packaging cell line may be any cell line that is capableof expressing retroviral proteins. Preferred packaging cell linesinclude 293 (ATCC CCL X), HeLa (ATCC CCL 2), D17 (ATCC CCL 183), MDCK(ATCC CCL 34), BHK (ATCC CCL-10) and Cf2Th (ATCC CRL 1430). The mostpreferable cell line is the 293 cell line.

The packaging cell line may stably express the necessary viral proteins.Such a packaging cell line is described, for example, in U.S. Pat. No.6,218,181. Alternatively a packaging cell line may be transientlytransfected with plasmids comprising nucleic acid that encodes thenecessary viral proteins.

In one embodiment a packaging cell line that stably expresses the viralproteins required for packaging the RNA genome is transfected with aplasmid comprising the viral construct described above.

In another embodiment a packaging cell line that does not stably expressthe necessary viral proteins is co-transfected with two or more plasmidsessentially as described in Yee et al. (Methods Cell. Biol. 43A, 99-112(1994)). One of the plasmids comprises the viral construct comprisingthe transgene. The other plasmid(s) comprises nucleic acid encoding theproteins necessary to allow the cells to produce functional virus thatis able to infect the desired host cell.

The packaging cell line may not express envelope gene products. In thiscase the packaging cell line will package the viral genome intoparticles that lack an envelope protein. As the envelope protein isresponsible, in part, for the host range of the viral particles, theviruses are preferably pseudotyped. Thus the packaging cell line ispreferably transfected with a plasmid comprising sequences encoding amembrane-associated protein that will permit entry of the virus into ahost cell. One of skill in the art will be able to choose theappropriate pseudotype for the host cell that is to be used. Forexample, in one embodiment the viruses are pseudotyped with thevesicular stomatitis virus envelope glycoprotein (VSVg). In addition toconferring a specific host range this pseudotype may permit the virus tobe concentrated to a very high titer. Viruses can alternatively bepseudotyped with ecotropic envelope proteins that limit infection to aspecific species, such as mice or birds. For example, in anotherembodiment, a mutant ecotropic envelope protein is used, such as theecotropic envelope protein 4.17 (Powell et al. Nature Biotechnology18(12):1279-1282 (2000)).

In the preferred embodiment a packaging cell line that does not stablyexpress viral proteins is transfected with the viral construct, a secondvector comprising the HIV-1 packaging vector with the env, nef, 5′LTR,3′LTR and vpu sequences deleted, and a third vector encoding an envelopeglycoprotein. Preferably the third vector encodes the VSVg envelopeglycoprotein.

In another embodiment of invention, RNA interference activity of thepackaging cells is suppressed to improve the production of recombinantvirus. This includes, without limitation, the use of cotransfection orstable transfection of constructs expressing siRNA molecules to inhibitDicer, an RNase III family member of ribonuclease which is essential forRNA interference (Hammond et al. Nat. Rev. Genet. 2:110-119 (2001)).

The recombinant virus is then preferably purified from the packagingcells, titered and diluted to the desired concentration.

Transgenic Animals

In order to make transgenic animals, an oocyte or one or more embryoniccells are infected with the recombinant virus produced as describedabove. One of skill in the art will recognize that the method ofinfection and the treatment of the cell following infection will dependupon the type of animal from which the cell is obtained. For example,mammalian cells are preferably implanted in a pseudopregnant femalefollowing infection while for the generation of transgenic birds orfish, the virus is preferably delivered to a laid egg and thusimplantation is not required.

While early methods of making transgenic animals required the cells tobe rapidly dividing, there is no such requirement in the methods of thepresent invention. Thus the cell may be contacted at any point indevelopment. In the preferred embodiment, a zygote is contacted with therecombinant virus.

The cells to be infected with the virus may be obtained by any methodknown in the art and appropriate for the specific species in which it isdesired to make a transgenic animal. For example, the recovery offertilized mouse oocytes is described in Hogan et al. (Manipulating theMouse Embryo: A Laboratory Manual. 2^(nd) ed. Cold Spring HarborLaboratory Press, NY (1994)). A method for obtaining fertilized ratoocytes is described in Armstrong et al. (Biol. Reprod. 39, 511-518(1998)).

It is not necessary that the cells be contacted after fertilization. Inone embodiment, the virus is delivered to unfertilized ova. Developmentmay then be initialized, for example by in vitro fertilization.

Delivery of the Virus

The virus may be delivered to the cell in any way that allows the virusto infect the cell. Preferably the virus is allowed to contact the cellmembrane. Two preferred methods of delivering the virus to mammaliancells, injection and direct contact, are described below.

Injection

In a first embodiment the virus is injected into the perivitelline spacebetween the zona pellucida and the cell membrane of a single-cellzygote. Preferably less than 50 picoliters of viral suspension isinjected, more preferably less than 25 picoliters and even morepreferably about 10 picoliters.

The virus is preferably present in a viral suspension and may beinjected by any method known in the art. The viral suspension ispreferably injected through a hydraulic injector. More preferably aglass micropipette is used to inject the virus. In one embodiment amicropipette is prepared by pulling borosilicate glass capillary on apipette puller. The tip is preferably opened and beveled toapproximately 10 μm. The lentiviral suspension may be loaded into themicropipette from the tip using gentle negative pressure.

In one embodiment the cell is stabilized with a holding pipette mountedon a micromanipulator, such as by gentle negative pressure against afire-polished pipette, and a second micromanipulator is used to directthe tip of a micropipette into the space between the zona pellucida andthe cell membrane, where the virus is injected.

Direct Contact

In another embodiment the zona pellucida is removed from the cell toproduce a denuded embryo and the cell membrane is contacted with thevirus. The zona pellucida may be removed by any method known in the art.Preferably it is removed by enzymatic treatment. For example, treatmentwith pronase may be used to remove the zona pellucida while the cellmembrane is kept intact. Alternatively, the cell may be placed in mediaat pH at which the zona pellucida dissolves while the cell membraneremains intact. For example the cell may be incubated in an acidicTyrode's solution at room temperature for several minutes. Once the zonapellucida is removed, any method that allows for the virus to contactthe cell membrane may be used. Preferably, the cell is incubated in asolution containing the virus. Even more preferably, the solution ismedia that facilitates survival of the cell.

In this embodiment, the cells are preferably contacted with the virus inculture plates. The virus may be suspended in media and added to thewells of a multi-well culture plate. The cells may then be plated in theindividual wells. The media containing the virus may be added prior tothe plating of the cells or after the cells have been plated. Preferablyindividual cells are incubated in approximately 10 μl of media. However,any amount of media may be used as long as an appropriate concentrationof virus in the media is maintained such that infection of the host celloccurs.

The cells are preferably incubated with the virus for a sufficientamount of time to allow the virus to infect the cells. Preferably thecells are incubated with virus for at least 1 hour, more preferably atleast 5 hours and even more preferably at least 10 hours.

Both the injection and direct contact embodiments may advantageously bescaled up to allow high throughput transgenesis. Because of the relativesimplicity of the injection technique, it is possible to inject manyembryos rapidly. For example, it is possible to inject more than 200fertilized oocytes in less than one hour. With regard to the directcontact embodiment, any number of embryos may be incubated in the viralsuspension simultaneously. This may be accomplished, for example, byplating the desired number of single-cell zygotes in multi-well tissueculture plates containing the virus suspended in media appropriate forthe survival and growth of the cells.

In both embodiments, any concentration of virus that is sufficient toinfect the cell may be used. Preferably the concentration is at least 1pfu/μl, more preferably at least 10 pfu/μl, even more preferably atleast 400 pfu/μl and even more preferably at least 1×10⁴ pfu/μl.

Following infection with the virus, the cells are preferably implantedin an animal. More preferably cells infected with the virus areimplanted in pseudopregnant animals of the same species from which theinfected cells were obtained. Methods of creating pseudo-pregnancy inanimals and implanting embryos are well known in the art and aredescribed, for example, in Hogan et al. (Manipulating the Mouse Embryo:A Laboratory Manual. 2^(nd) ed. Cold Spring Harbor Laboratory Press, NY(1994)).

In the preferred embodiment early stage embryos (approximately 0-2.5days p.c.) still with an intact zona pellucida are transferred to theoviduct of timed pseudopregnant female (preferably 0.5 days p.c.), whileembryos that have reached the blastocyst stage are transferred to theuterus of timed pseudopregnant females (preferably 2.5 days p.c.).Denuded embryos are preferably cultured in vitro until they reach themorula or blastocyst stage (48 to 72 hours in culture), and are thenimplanted into appropriately timed pseudopregnant females.

The embryos and resulting animals may be analyzed, for example forintegration of the transgene, the number of copies of the transgene thatintegrated, the location of the integration, the ability to transmit thetransgene to progeny and expression of the transgene. Such analysis maybe carried out at any time and may be carried out by any methods knownin the art. Standard techniques are described, for example, in Hogan etal. (supra).

The methods of infecting cells disclosed above do not depend uponspecies-specific characteristics of the cells. As a result, they arereadily extended to all mammalian species.

Initial experiments with mice indicate that of those animals thatdevelop to full term, 80-90% carried at least one copy of the transgeneand that, of these, approximately 85% express the gene of interest. Ofthe transgenic animals about 25% carry only 1 or 2 copies of thetransgene. The highest number of proviral insertions observed was about30. Of the animals that carried only 1 or 2 copies of the transgene,about 80% expressed the gene of interest.

As discussed above, the modified retrovirus can be pseudotyped to conferupon it a broad host range. One of skill in the art would also be awareof appropriate internal promoters to achieve the desired expression of agene of interest in a particular animal species. Thus, one of skill inthe art will be able to modify the method of infecting cells to createtransgenic animals of any species.

In one embodiment, transgenic birds are created by delivering a modifiedretrovirus, as described above, to the primordial germ cells of earlystage avian embryos. Freshly laid eggs are obtained and placed in atemperature controlled, humidified incubator. Preferably, the embryonicblastodisc in the egg is gradually rotated to lie on top of the yolk.This may be accomplished by any method known in the art, such as bygently rocking the egg regularly, preferably every 15 minutes.Approximately 36 hours later, the modified retrovirus is delivered intothe space between the embryonic disk and the perivitelline membrane.Preferably about 50 nL of viral solution is delivered, more preferablyabout 100 nL of viral solution is delivered, and even more preferablyabout 200 nL of viral solution is delivered. The viral solution may bedelivered by any method known in the art for delivering compositions tothe inside of an egg. In the preferred embodiment a window is opened inthe shell, the viral solution is injected through the window and theshell window is closed. The eggs are preferably incubated untilhatching. The eggs will hatch after approximately 20 days, dependingupon the particular avian species from which they are obtained. Hatchedchicks are preferably raised to sexual maturity and mated. Thetransgenic offspring of the founder animals may be identified by anymethod known in the art, such as Southern blot, PCR and expressionanalysis.

In another embodiment, transgenic fish are created by delivering themodified retrovirus, described above, to single-cell fish embryos.Fertilized fish eggs are collected by any method known in the art. Themodified retrovirus is then preferably delivered to the space betweenthe chorion and the cell membrane. This may be accomplished, forexample, by loading the modified retrovirus in solution into a glasspipette. The pipette may then be used to pierce the chorion membrane anddeliver the viral suspension. Preferably about 50 nL of viral solutionis delivered, more preferably about 100 nL of viral solution isdelivered, and even more preferably about 200 nL of viral solution isdelivered. Injected embryos are preferably returned to atemperature-controlled water tank and allowed to mature. At sexualmaturity the founder fish are preferably mated and their progenyanalyzed for the presence of the transgene by any method known in theart.

As mentioned above, the methods of the present invention will also proveuseful in techniques for identifying genes that are involved in specificbiological processes, such as gene trap assays and large-scalemutagenesis screens. Such methods are described in the application60/322,031 filed on Sep. 13, 2001 and U.S. provisional patentapplication 60/347,782 filed on Jan. 9, 2002.

Down-Regulating Gene Expression in a Target Cell

The methods described herein allow the expression of RNA molecules incells, and are particularly suited to the expression of small RNAmolecules, which can not be readily expressed from a Pol II promoter.According to a preferred embodiment of the invention, an RNA molecule isexpressed within a cell in order to down-regulate the expression of atarget gene. The ability to down-regulate a target gene has manytherapeutic and research applications, including identifying thebiological functions of particular genes. Using the techniques andcompositions of the invention, it will be possible to knock-down (ordown-regulate) the expression of a large number of genes, both in cellculture and in mammalian organisms.

In preferred embodiments of the invention, an RNA expression cassettecomprises a Pol III promoter and an RNA coding region. The RNA codingregion preferably encodes an RNA molecule that is capable ofdown-regulating the expression of a particular gene or genes. The RNAmolecule encoded can, for example, be complementary to the sequence ofan RNA molecule encoding a gene to be down-regulated. In such anembodiment, the RNA molecule preferably acts through an antisensemechanism.

A more preferred embodiment involves the expression of a double-strandedRNA complex, or an RNA molecule having a stem-loop or a so-called“hairpin” structure. As used herein, the term “RNA duplex” refers to thedouble stranded regions of both the RNA complex and the double-strandedregion of the hairpin or stem-lop structure.

Double stranded RNA has been shown to inhibit gene expression of geneshaving a complementary sequence through a process termed RNAinterference or suppression (see, for example, Hammond et al. Nat. Rev.Genet. 2:110-119 (2001)).

According to the invention, an RNA duplex or siRNA corresponding to aregion of a gene to be down-regulated is expressed in the cell. The RNAduplex is substantially identical (typically at least about 80%identical, more preferably at least about 90% identical) in sequence tothe sequence of the gene targeted for down regulation. siRNA duplexesare described, for example, in Bummelkamp et al. Science 296:550-553(2202), Caplen et al. Proc. Natl. Acad. Sci. USA 98:9742-9747 (2001) andPaddison et al. Genes & Devel. 16:948-958 (2002).

The RNA duplex is generally at least about 15 nucleotides in length andis preferably about 15 to about 30 nucleotides in length. However, asignificantly longer RNA duplex can be used effectively in someorganisms. In a more preferred embodiment, the RNA duplex is betweenabout 19 and 22 nucleotides in length. The RNA duplex is preferablyidentical to the target nucleotide sequence over this region.

When the gene to be down regulated is in a family of highly conservedgenes, the sequence of the duplex region can be chosen with the aid ofsequence comparison to target only the desired gene. On the other hand,if there is sufficient identity among a family of homologous geneswithin an organism, a duplex region can be designed that would downregulate a plurality of genes simultaneously.

The duplex RNA can be expressed in a cell from a single retroviralconstruct. In the preferred embodiment, a single RNA coding region inthe construct is a serves as a template for the expression of aself-complementary hairpin RNA, comprising a sense region, a loop regionand an antisense region. This embodiment is illustrated in FIG. 2, whichshows a schematic view of an RNA expression cassette having an RNA PolIII promoter 100 operatively linked to an RNA coding region, having asense region 110, a loop region 120, an antisense region 130 and aterminator region 140. The sense 110 and antisense 130 regions are eachpreferably about 15 to about 30 nucleotides in length. The loop region120 preferably is about 2 to about 15 nucleotides in length, morepreferably from about 4 to about 9 nucleotides in length. Followingexpression the sense and antisense regions form a duplex.

In another embodiment, the retroviral construct comprises two RNA codingregions. The first coding region is a template for the expression of afirst RNA and the second coding region is a template for the expressionof a second RNA. Following expression, the first and second RNA's form aduplex. The retroviral construct preferably also comprises a first PolIII promoter operably linked to the first RNA coding region and a secondPol III promoter operably linked to the second RNA coding region. Thisembodiment is illustrated in FIG. 3, which shows a schematic view of anRNA expression cassette having an RNA Polymerase III promoter 100 linkedto a first RNA coding region 110 and a first terminator sequence 140 anda second RNA polymerase III promoter 105 linked to a second RNA codingregion 115 and a second terminator 145.

In yet another embodiment of the invention, the retroviral constructcomprises a first RNA Pol III promoter operably linked to a first RNAcoding region, and a second RNA Pol III promoter operably linked to thesame first RNA coding region in the opposite direction, such thatexpression of the RNA coding region from the first RNA Pol III promoterresults in a synthesis of a first RNA molecule as the sense strand andexpression of the RNA coding region from the second RNA Pol III promoterresults in synthesis of a second RNA molecule as an antisense strandthat is substantially complementary to the first RNA molecule. In onesuch embodiment, both RNA Polymerase III promoters are separated fromthe RNA coding region by termination sequences, preferably terminationsequences having five consecutive T residues. FIG. 4 shows a schematicview of an RNA expression cassette having a first RNA Polymerase IIIpromoter 100 linked to an RNA coding region 110 and a first terminatorsequence 145. The expression cassette has a second RNA polymerase IIIpromoter 105 linked to the RNA coding region 115, the same sequence as110 in reverse, and a second terminator 140.

In further embodiments an RNA duplex is expressed using two or moreretroviral constructs. In one embodiment, a first retroviral constructis used that directs the expression of a first RNA and a secondretroviral construct is used that directs expression of a second RNAthat is complementary to the first. Following expression the first andsecond RNAs form a duplex region. It is preferred, however, that theentire duplex region is introduced using retroviral particles derivedfrom a single retroviral construct. As discussed above, severalstrategies for expressing a duplex RNA from a single viral construct areshown in FIGS. 2-4.

The RNA duplexes may be flanked by single stranded regions on one orboth sides of the duplex. For example, in the case of the hairpin, thesingle stranded loop region would connect the duplex region at one end.

The RNA coding region is generally operatively linked to a terminatorsequence. The pol III terminators preferably comprise of stretches of 4or more thymidine (“T”) residues. In a preferred embodiment, a clusterof 5 consecutive Ts is linked immediately downstream of the RNA codingregion to serve as the terminator. In such a construct pol IIItranscription is terminated at the second or third T of the DNAtemplate, and thus only 2 to 3 uridine (“U”) residues are added to the3′ end of the coding sequence.

The sequence of the RNA coding region, and thus the sequence of the RNAduplex, preferably is chosen to be complementary to the sequence of agene whose expression is to be downregulated in a cell or organism. Thedegree of down regulation achieved with a given RNA duplex sequence fora given target gene will vary by sequence. One of skill in the art willbe able to readily identify an effective sequence. For example, in orderto maximize the amount of suppression in a transgenic animal, a numberof sequences can be tested for their efficacy in cell culture prior togenerating a transgenic animal.

The methods of the present invention will find great commercialapplication, for example in biotechnology, medicine and agriculture. Forexample, in agriculture the described methods may be used to conferdisease resistance by expressing in a cell or organism an siRNA thatspecifically down-regulates the expression of a gene associated with apathogen or disease state. In biotechnology, the ability to rapidlydevelop large numbers of transgenic animals with desired modulation ofspecific genes will allow for the analysis of gene function and theevaluation of compounds that potentially modulate gene expression,protein function, and are useful in treating a disease or disorder. Inparticular, by observing the effect of down-regulating specific genes intransgenic animals, the biological function of those genes may bedetermined. In medicine the methods of the invention may be used totreat patients suffering from particular diseases or disorders, such asHIV, or to confer immunity or resistance to particular pathogens. Forexample, specific cells may be infected in vivo or ex vivo withrecombinant retrovirus encoding an siRNA that down-regulates theactivity of a gene whose activity is associated with a particulardisease or disorder.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and fall within the scope of theappended claims.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES Example 1

An lentiviral construct was constructed by insertion of an siRNAexpression cassette into the PacI site of HC-FUGW vector (FIG. 5; SEQ IDNO: 2). The siRNA was designed to down-regulate expression of the lacZgene. The HC-FUGW vector comprised a GFP marker gene operably linked tothe human Ubiquitin promoter. The GFP marker was useful for trackingtransduction events. The vector also comprised an HIV DNA Flap elementto improve the virus titers, and the WRE for high level expression ofviral genes. The siRNA expression cassette was composed of a pol IIIpromoter and a small hairpin RNA coding region followed by a pol IIIterminator site. The pol III promoter (SEQ ID NO:3) was derived from the−240 to −9 region of human H1-RNA promoter and was cloned as an Eco R1fragment by PCR amplification from HEK293 genomic DNA. The pol IIIpromoter was connected to the downstream RNA coding region by a 7 basepair linker sequence to ensure that the transcription was preciselyinitiated at the first nucleotide of the RNA coding sequence. The smallhairpin RNA coding region comprised a 19 nt sequence corresponding tothe 1900-1918 region of the sense strand of the bacterialbeta-galactosidase (lacZ) gene coding sequence and the 19 nt perfectreverse complementary sequence separated by a 9 nt loop region. Theterminator was comprised of 5 consecutive thymidine residues linkedimmediately downstream of the RNA coding sequence. The sequence of thehairpin siRNA is shown in SEQ ID NO: 1.

Example 2

Transduction of cultured mammalian cells with retrovirus derived fromthe retroviral construct described in Example 1 was achieved (FIG. 6).The retroviral vector encoding a small hairpin RNA molecule described inExample 1, was used to transfect cultured mammalian cells that expresslacZ. A profound decrease in the expression of the lacZ was observed.

The lacZ siRNA virus was produced by cotransfection of the retroviralvector, a helper virus plasmid and VSVg expression plasmid in HEK293cells. The virus particles were harvested from the cell culturesupernatants and concentrated by ultracentrifugation. The concentratedvirus preparations were used to infect either mouse embryonicfibroblasts (MEF) or HEK293 cells which harbor both lacZ and fireflyluciferase (Luc) reporter genes. Infection was monitored by the GFPsignal which is expressed from the marker gene cassette of the viralvector. Under the conditions of this experiment, >98% of the test cellswere GPF+ and thus were successfully transduced. The expression levelsof lacZ and Luc reporter genes were measured by chemiluminescent assaysusing commercially available kits (lacZ assay kit from Roche and Lucfrom Promega). The lacZ siRNA virus only inhibited the expression oflacZ but not Luc. The specific inhibition was determined by the ratio oflacZ activity to Luc activity. The lacZ/Luc ratio of the uninfectedparental cells was arbitrarily set to 1 and the values of the infectedcells were calculated accordingly. As shown in FIG. 6, transfection withthe virus resulted in dramatic reduction in the amount of expression ofthe lacZ gene in both MEK and HEK293 cells.

A tet-iducible lacZ siRNA lentiviral vector was also prepared asillustrated in FIG. 8. A Tet repressor gene (TetR; SEQ ID NO: 5) wasplaced the under the control of the human UbiquitinC promoter so thatits expression could be monitored by the downstream GFP marker. Theanti-lacZ siRNA cassette was driven by a Tet-inducible pol III promoterderived from human U6-promoter (−328 to +1) containing a single TetRbinding site (TetO1) between the PSE and TATA box (SEQ ID NO: 4). TheTetR coding sequence was PCR amplified from genomic DNA from the TOP10strain of E. coli adn cloned into a modified version of FUIGW as aBgl2-EcoR1 fragment. In the absence of tetracycline, TetR binds to thepromoter and its expression is repressed. Upon the addition oftetracycline, TetR is moved from the promoter and transcription starts.

The Tet-inducible siRNA expression cassette was able to regulate geneexpression in response to Doxycycline treatment. Virus was prepared fromthe retroviral construct carrying the Tet-inducible lacZ-siRNA cassetteand a Tet repressor under the control of a UbiquitinC promoter and usedto transduce HEK293 cells expressing both lacZ and luciferase(293Z+Luc). The transduced cells were treated with 10 ug/ml Doxycycline(Plus Dox) for 48 hr or without the Doxycycline treatment as a control(No Dox). LacZ and luciferase activities were measured as described inthe previous figures. The relative suppression activity is calculated asthe ratio of lacZ versus luciferase and No Dox control was arbitrarilyset to 1. As can be seen in FIG. 9, in the presence of doxycyclinesuppression of lacZ activity was significantly enhanced.

Example 3

This example demonstrates the generation of transgenic animals thatexpress an siRNA molecule encoded by a lentiviral vector. The expressionof the lacZ specific siRNA described in Example 1 resulted in extensivesuppression of lacZ activity in ROSA26+/− mice.

ROSA26+/− mice carry one copy of a ubiquitously expressed lacZ reportergene. The lacZ siRNA virus preparations described in Example 2 were usedfor perivitelline injection of ROSA26+/− single cell embryos obtainedfrom hormone primed C57Bl/6 female donors×ROSA26+/+ stud males. Theinjected single cell embryos were subsequently transferred into theoviduct of timed pseudopregnant female recipients. Embryonic day 15.5 to17.5 (E15.5-17.5) fetuses were recovered from the surrogate mothers.Successful transgenesis was scored by positive GFP signal observed withthe fetuses under fluorescent microscope. Protein extracts prepared fromthe limb tissues of the fetuses were used for the LacZ chemiluminescentassay according to the manufacturer's instruction (Roche), and proteinconcentrations of the tissue extracts were determined by the Bradfordassay (BioRad). The lacZ expression levels were expressed as light units(LU) per ug of proteins (LU/ug). The E15.5-17.5 fetuses from the timedmating of C57Bl/6 females×ROSA26+/+ males and C57Bl/6 females×C57Bl/6males were served as the positive and negative controls respectively.The results are shown in FIG. 7. Animals G1-G4 (those treated derivedfrom embroys infected with the virus comprising the siRNA construct)showed markedly decreased expression of the lacZ gene as compared withuntreated control animals.

What is claimed is:
 1. A retroviral construct for the expression of anRNA in a cell, the retroviral construct comprising: a nucleic acidhaving the R and U5 sequences from a 5′ lentiviral long terminal repeat(LTR); a self-inactivating lentiviral 3′ LTR; and a first RNA PolymeraseIII promoter configured to operably linked to a first RNA coding regionencoding a first desired RNA, wherein the first RNA Polymerase IIIpromoter is located between the 5′ LTR and the 3′ LTR.
 2. The retroviralconstruct of claim 1, wherein the first desired RNA is a small RNAmolecule with regulatory function.
 3. The retroviral construct of claim1, wherein the first desired RNA is a RNA marker, an antisense RNA, aribozymes, or a small interfering RNA (siRNA).
 4. The retroviralconstruct of claim 1, wherein the RNA Polymerase III promoter isinducible.
 5. The retroviral construct of claim 4, further comprising asecond RNA Polymerase III promoter operably linked to a second RNAcoding region encoding a second desired RNA.
 6. The retroviral constructof claim 5, wherein the second desired RNA is substantiallycomplementary to the first desired RNA.
 7. The retroviral construct ofclaim 1, wherein the first RNA coding region is followed by at least onetermination sequence.
 8. The retroviral construct of claim 1, whereinthe first desired RNA is a self-complementary RNA molecule having asense region, an antisense region and a loop region.
 9. The retroviralconstruct of claim 8, wherein the loop region is about 2 to about 10nucleotides in length.
 10. The retroviral construct of claim 8, whereinthe sense region and the antisense region are between about 15 and about30 nucleotides in length.
 11. The retroviral construct of claim 1,wherein expression of the first desired RNA results in the downregulation of a target gene.
 12. The retroviral construct of claim 1,wherein the 5′ LTR sequence is from HIV.
 13. The retroviral construct ofclaim 1, wherein the self-inactivating 3′ LTR is a modified HIV 3′ LTR.14. The retroviral construct of claim 1, further comprising a gene ofinterest.
 15. The retroviral construct of claim 14, wherein the gene ofinterest is operably linked with a Polymerase II promoter.
 16. Theretroviral construct of claim 15, wherein the Polymerase II promoter isa CMV promoter, or a ubiquitous promoter, or a tissue specific promoter.17. The retroviral construct of claim 16, wherein the ubiquitouspromoter is selected from the group consisting of ubiquitin promoter,cytomegalovirus (CMV) promoter, β-actin promoter, and PGK promoter. 18.The retroviral construct of claim 16, wherein the tissue specificpromoter is selected from the group consisting of 1ck promoter, myogeninpromoter and thy1 promoter.
 19. The retroviral construct of claim 14,wherein the gene of interest is a reporter gene.
 20. The retroviralconstruct of claim 1, further comprising an enhancer sequence.